Construction Of Functional DNA Nano Self-assemblies For The Application In Cancer Diagnosis And Therapy

Cancer is a conundrum in modern medicine and has become a serious threat to human health and life.In general,the occurrence of cancer is closely associated with the expression of certain specific biomarkers.However,the low expression abundance of these biomarkers in cells often causes the unsatisfactory results by using the conventional detection means.Therefore,the development of a detection probe with high sensitivity and signal amplification function is of great significance for the early detection of cancer-related biomarkers.In terms of cancer therapy,a series of organic or inorganic nanomaterials have been developed for the construction of drug delivery systems.However,most nanomaterials are still challenged by obvious cytotoxicity,structural instability and non-specificity.In addition,the complicated preparation process also largely limits their clinical application.In recent years,DNA nanomaterials have been widely used for biological analysis and drug delivery due to their excellent biocompatibility,programmable,and easy modification.On this basis,this dissertation intends to use DNA nanotechnology to build highly sensitive detection platform,meanwhile,develop new multifunctional DNA nano self-assemblies for combined anti-cancer therapy and ultimately achieve the integration of cancer diagnosis and therapy.The main research works are summarized as follows:1.Cascade Amplification-Mediated In Situ Hot-Spot Assembly for Micro RNA Detection and Molecular Logic Gate Operations: Micro RNAs(mi RNAs)are usually detected by combination of a single signal amplification strategy and various detection techniques.However,these methods are often limited by their low sensitivity and selectivity,leading to the detection results are undesirability.In this work,we have fabricated a cascade signal amplification label-free electrochemical biosensor through integrating duplexspecific nuclease(DSN)-assisted target recycling with catalytic hairpin assembly(CHA)reaction for the highly sensitive detection of mi RNA-141.The biosensor possesses excellent repeatability and stability,with the detection limit of 25.1 a M and the linear range of 0.1 f M-10 n M.Meanwhile,it can accurately identify single-base mismatched mi RNA-141.Finally,this biosensor is successfully used for the quantitative detection of mi RNA-141 in MDAMB-231 cells.On this basis,this work also constructed two logic gates including AND gate and INH gate,in which,AND gate can further realize the preliminary qualitative analysis of mi RNAs in cancer cells.Therefore,the biosensor constructed in this work has great potential applications in mi RNA-related clinical diagnostics and biochemical research.2.Multifunctional DNA Polycatenane Nanocarriers for Synergistic Targeted Therapy of Multidrug-Resistant Human Leukemia: Chemotherapy,as one of the principal modalities for cancer therapy,often leads to the unsatisfactory therapeutic effect due to its inefficient delivery,serious side effects and especially the multidrug resistance resulting from long-term use.On the other hand,although there have been reported numerous studies about DNA nanostructures as drug nanocarriers,the low self-assembly efficiency of DNA nanostructures leads to the complex structural components,and the current purification approaches(including centrifugation,dialysis and gel electrophoresis)always result in the structure destruction and samples contamination.Herein,we construct a multifunctional aptamer-tethered DNA polycatenanes(Apt DPCs)with high purity through the recyclable magnetic separation method,and combine chemotherapy and gene therapy to achieve targeted synergistic therapy of multidrug-resistant human leukemia.When the Doxloaded Apt DPCs are specifically internalized into the multidrug-resistant human leukemia cells,Dox could rapidly release under the acidic environment of endosomes,and gradually diffusing into the nucleus for inducing tumor cell apoptosis.Meanwhile,the antisense DNA can specifically hybridize with the MDR1 m RNAs to suppress the expression of P-gp and protected Dox from efflux,thereby enhancing the chemosensitivity of MDR cancer cells to drugs.In vitro and in vivo results have confirmed that Apt DPCs can not only achieve targeted drug delivery,but also significantly enhance chemotherapeutic efficacy of MDR tumor via the synergistic gene therapy,which opens a new avenue for the development of MDR cancertargeted nanotherapeutic platforms and presents tremendous significance in clinical fields.3.Endogenous m RNA Triggered DNA-Au Nanomachine for in Situ Imaging and Targeted Multimodal Synergistic Cancer Therapy: Accurate diagnosis and therapy of cancers are of great significance to improve the cure rate of patients.However,the design of versatile tumor nanotheranostic platforms that integrate both diagnostic and therapeutic functions have always been an intractable challenge.Herein,we propose a multifunctional DNA nanomachine(Apt-DNA-Au)based on the entropy-driven DNA strand displacement reaction for in situ imaging of cancer-related biomarkers and multimodal synergistic antitumor therapy.The designed nanomachine makes Au NPs as carrier,and the three component DNA hybrid,the single-stranded fuel DNA and the MUC1 aptamer,all of which are immobilized on its surface via Au-S covalent bond.When the Apt-DNA-Au nanomachine is specifically internalized into the MCF-7 cells,under the trigger of TK1 m RNA,the Cy5 fluorescence is recovered via DNA cascades strand displacement reaction and used for in situ imaging of target,which thus facilitates the real-time monitoring of the dynamic changes of tumor during therapy.In this process,the conformation changes of the nanomachine and the stimulation of acidic environment in tumor sites facilitate the precisely controlled release of Dox for chemotherapy.Meanwhile,the antisense DNA is liberated to down-regulated the expression of survivin for gene therapy.Moreover,under the external 660 nm laser irradiation,the aggregated Au NPs and the photosensitizer loaded in G-quadruplex can generate hyperthermia and reactive oxygen species(ROS)for photothermal therapy(PTT)and photodynamic therapy(PDT),respectively.In vitro and in vivo studies confirm that the proposed Apt-DNA-Au nanomachine not only achieves synergistic therapy of breast cancer in combination of chemotherapy,gene regulation,PTT and PDT,but also contributes to accurate diagnosis of tumors and imaging-guided cancer therapy,which provides new ideas for the construction of theranostic nanoplatform.